CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector against inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target GBM. T immunoglobulin mucin family member 3 (TIM3), receptor expressed on cells, has been suggested as marker of dysfunctional cells. We established TIM3 knockout using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating exon 2- or 5-targeting guide RNA- Cas9 complexes (RNPs) inhibited expression with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted intended genome sites. The other checkpoint receptors, i.e., programmed death 1 (PD1), Lymphocyte-activation gene (LAG3), immunoreceptor Ig ITIM domains (TIGIT), TACTILE (CD96) were unchanged Real time growth revealed enhanced cell–mediated inhibition These results demonstrated human mediated cytotoxicity Future, CRISPR-Cas9 may prove to immunotherapeutic alternative patient